Withania somnifera Dunal (WS) of family Solanaceae, known as Ashwagandha in Ayurveda, the ancient Hindu system of medicine, has been in use for more than 2500 years. The roots of the plant were used in rasayana formulations, a group of plant-derived drugs that are reputed to promote health and longevity by augmenting body's defense mechanisms against disease, arresting the aging process, revitalizing the body in debilitated conditions, increasing the capability of the individual to resist adverse environmental factors and creating a sense of mental well-being. M. A. Weiner, J. Weiner, Ashwagandha (Indian Ginseng), pp. 70-72, Herbs that Heal (Mill Valley, Calif.: Quantum Books, 1994).
Several earlier investigations have indicated that WS has a profile of activity that is consonant with putative anti-stress and antioxidant activity. WS, or its major active principles, have anti-inflammatory activity, antitumor and radio-sensitizing actions and have annulled cyclophosphamide toxicity. Likewise, the active principles of WS, comprising sitoindosides VII-X and withaferin-A, have been shown to have significant antistress activity against acute and chronic models of experimental stress, immunomodulatory actions, inhibition of cognitive deficits in animal models of Alzheimer's disease, antioxidant activity in rat brain areas, and anxiolytic-antidepressant action in rats (S. K. Bhattacharya, et al., “Antistress activities of sitoindosides VII and VIII, new acyl sterylglucosides from Withania somnifera,” Phytother. Res. (1987) 1:32-37; S. K. Bhattacharya, Muruganandam A. V., “Adaptogenic activity of Withania somnifera: an experimental study using a rat model of chronic stress,” Pharmacol Biochem. and Behavior (2003) 75: 547-555; S. Ghosal, et al., “Immunomodulatory and CNS effects of sitoindosides IX and X, two new glycowithanolides from Withania somnifera,” Phytother. Res. (1989) 3: 201-206; S. K. Bhattacharya, et al., “Effect of Trasina, an Ayurvedic herbal formulation on experimental models of Alzheimer's disease and central cholinergic markers in rats,” J. Altern. Complement. Med. (1997) 3:327-336; S. K. Bhattacharya, et al., “Antioxidant activity of glycowithanolides from Withania somnifera,” Indian J. Exp. Biol. (1997) 35: 236-239; and S. K. Bhattacharya, et al., “Anxiolytic-antidepressant activity of Withania somnifera glycowithanolides: an experimental study,” Phytomed. (2000) 7: 463-469). WS root extract and its constituents, withanolides and withanosides, were also reported to possess anti-acetylcholinesterase activity and dementia thwarting activity (M. I. Choudhary, et al. “Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties,” Biochem. and Biophys. Res. Comm. (2005) 334: 276-287).
Although significant numbers of studies have been carried out on WS and its bioactives, many of them are centered on withasteroids, namely, withanolides and their glycosides. Recently, a new class of compounds, withanamides, from the fruit of WS, having antioxidant, anti-inflammatory and β-amyloid protecting activities were reported (B. Jayaprakasam et al., “Potent lipid peroxidation inhibitors from Withania somnifera fruits,” Tetrahedron (2004) 60: 3109-3121; and B. Jayaprakasam et al., “Withanamides in Withania somnifera fruit protect PC-12 cells from beta-amyloid responsible for Alzheimer's disease,” Phytother. Res. (2010) 24:859-63). Acetylcholinesterase inhibitors (AChEIs) are an important class of compounds which are indicated for the management of mild to moderate Alzheimer's dementia. Alzheimer's disease is associated with significant losses in cholinergic neurons and decreased concentrations of the neurotransmitter, acetylcholine, which is significantly involved in learning and memory processes. AChEIs exert pharmacologic effects by increasing availability of intrasynaptic acetylcholine in the presence of intact cholinergic neurons. There are a few synthetic medicines, e.g., Tacrine, Donepezil, Galantamine, and the natural product-based Rivastigmine that are currently being used for treatment of cognitive dysfunction and memory loss associated with Alzheimer's disease. These compounds, however, are not free from certain adverse effects including gastrointestinal disturbances and problems associated with bioavailability. The clinical usefulness of AChEIs has been limited by either an extremely short or an excessively long half-life, hepatotoxicity, and severe peripheral cholinergic side effects.
Two studies reported on the AChE activity of WS, although they were confined to the total extract and withanolides only. (M. I. Choudhary, et al., “Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties,” Biochemical and Biophysical Res. Comm. (2005) 334: 276-287; and S. Khattak, et al., “In vitro enzyme inhibition activities of crude ethanolic extracts derived from medicinal plants of Pakistan,” Nat. Prod. Res. (2005) 19:567-571.)
WS was shown to possess learning and memory improvement activity in various animal models by different investigators. One study investigated the active principles of WS, consisting of equimolar amounts of sitoindosides VII-X and withaferin A, for putative nootropic activity in an experimentally validated Alzheimer's disease model. The syndrome was induced by ibotenic acid lesioning of the nucleus basalis magnocellularis (NBM) in rats. WS significantly reversed both ibotenic acid-induced cognitive deficits and the reduction in cholinergic markers after 2 weeks of treatment. The findings validate the medharasayan (promoter of learning and memory) effect of WS, as has been reported in Ayurveda. (S. K. Bhattacharya, et al., “Effects of glycowithanolides from Withania somnifera on an animal model of Alzheimer's disease and perturbed central cholinergic markers of cognition in rats,” Phytother. Res. (1995) 9: 110-113.)
In another study, sitoindosides VII-X, and withaferin-A, were isolated from aqueous methanol extract from the roots of cultivated varieties of WS to attenuate cerebral functional deficits, including amnesia, in geriatric patients. Systemic application of the defined extract from WS, however, led to differential effects on AChE activity in basal forebrain nuclei; slightly enhanced AChE activity was found in the lateral septum and globus pallidus, whereas in the vertical diagonal band AChE activity was reduced following treatment with sitoindosides VII-X and withaferin-A. These changes were accompanied by enhanced M1-muscarinic cholinergic receptor binding in lateral and medial septum as well as in frontal cortices, whereas the M2-muscarinic receptor binding sites were increased in a number of cortical regions including cingulate, frontal, piriform, parietal and retrosplenial cortex. Treatment with the defined extract from WS affected neither GABA and benzodiazepine receptor binding nor NMDA and AMPA glutamate receptor subtypes in any of the cortical or subcortical regions studied. The data suggest that the defined extract from WS affect preferentially events in the cortical and basal forebrain cholinergic signal transduction cascade. The drug-induced increase in cortical muscarinic acetylcholine receptor capacity might partly explain the cognition-enhancing and memory-improving effects of extracts from WS observed in animals and humans. (R. Schliebs, et al., “Systemic administration of defined extracts from Withania somnifera (Indian Ginseng) and Shilajit differentially affects cholinergic but not glutamatergic and GABAergic markers in rat brain,” Neurochem. Int. (1997) 30:181-190.)
The anxiolytic and the anti-depressant effects of glycowithanolides from WS were compared with those elicited by the anti-anxiety drug Lorazepam and by the antidepressant, Imipramine. Glycowithanolides induced an anxiolytic effect, comparable to that produced by Lorazepam, in the elevated plus-maze, social interaction, and feeding latency in an unfamiliar environment tests. Further, both the glycowithanolides and Lorazepam reduced rat brain levels of Tribulin, an endocoid marker of clinical anxiety, when the levels were increased following administration of the anxiogenic agent, pentylenetetrazole. Glycowithanolides also exhibited an antidepressant effect, comparable to that induced by Imipramine, in the forced swim-induced behavioral despair and learned helplessness tests. This investigation supports the use of WS as a mood stabilizer in clinical conditions of anxiety and depression in Ayurveda, and in other treatment paradigms. (S. K. Bhattacharya, et al., “Anxiolytic-antidepressant activity of WS glycowithanolides: an experimental study,” Phytomed. (2000) 7: 463-469.)
WS root extract administration improved retention of a passive avoidance task in a step-down paradigm in mice. WS also reversed the scopolamine-induced disruption of acquisition and retention and attenuated the amnesia produced by acute treatment with electroconvulsive shock. On the elevated plus-maze, Ashwagandha reversed the scopolamine-induced delay in transfer latency on day 1. On the basis of these findings, it is suggested that Ashwagandha exhibits a nootropic-like effects in naive and amnesic mice. In another study, six compounds were isolated from the methanol extract of WS roots which enhanced neurite outgrowth in human neuroblastoma SH-SY5Y cells. That study also reported that in withanolide A-treated cells, the length of NF—H-positive process was significantly increased compared to vehicle-treated cells, whereas, the length of MAP2-positive process was increased by withanolides. (J. N. Dhuley, “Nootropic-like effect of Ashwagandha (WS L.) in mice,” Phytother. Res. (2001) 15: 524-5288; and T. Kuboyama, et al., “Axon or dendrite-predominant outgrowth induced by constituents from Aswagandha,” Neuroreport. (2002) 13: 1715-1717.)
Bhattacharya and Muruganandam A. V., cited above, investigated the adaptogenic activity of a standardized extract of WS roots against a rat model of chronic stress (CS). The stress procedure was mild, unpredictable footshock, administered once daily for 21 days to adult male Wistar rats. CS induced significant hyperglycaemia, glucose intolerance, increase in plasma corticosterone levels, gastric ulcerations, male sexual dysfunction, cognitive deficits, immunosuppression and mental depression. These CS induced perturbations were attenuated by WS extract administered 1 hour before footshock for 21 days. The results indicate that WS has significant antistress and adaptogenic activity.
It was also reported that withanolides 1-3, and 4 and 5 isolated from Ajuga bracteosa and WS, respectively, inhibited acetylcholinesterase and butyrylcholinesterase enzymes in a concentration-dependent fashion. It was suggested that the cholinesterase inhibitory potential along with calcium antagonistic ability and safe profile in human neutrophil viability assay could make withanolides 1-5 possible drug candidates for further study to treat Alzheimer's disease and associated problems. It was also reported that some active constituents of WS such as withanolide A, withanoside IV and withanoside VI could improve Amyloid-β (25-35)-induced memory impairment, neuronal atrophy and synaptic loss in the cerebral cortex and the hippocampus. (M. I. Choudhary, et al., “Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties,” Biochem. and Biophys. Res. Comm. (2005) 334: 276-287; and T. Chihiro, et al., “Scientific basis for the anti-dementia drugs of constituents from Ashwagandha (WS),” J. Trad. Med. (2005) 22:176-182.)
Anxiolytic and anti-depressant activities of WS root extract in social isolation induced behavior such as anxiety and depression in rats, has been reported. (G. L. Gupta, et al., “Protective Effect of WS Dunal Root Extract against Protracted Social Isolation Induced Behavior in Rats,” Indian J. Physiol. Pharmacol. (2007) 51: 345-353.)
Oral administration of WS extract exerts protective effect and attenuates AChE inhibition and cognitive impairment caused by sub-chronic exposure to Propoxur, which blocks the production and action of acetylcholinesterase. (C. S. Yadav, et al., “Propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function: attenuation by Withania somnifera,” Indian J. Biochem. Biophys. (2010) 47:117-20.)
As discussed above, several bioactive principles of WS have been isolated and their antioxidant, anti-stess, anxiolytic and anti-cholinesterase activities have been extensively studied. The drugs commonly used as anxiolytics, for example the benzodiazepines, and drugs used for treating Alzheimer's disease can have severe side effects. Thus there is a need and a desire for a better class of drugs without adverse side effects. The present invention describes the isolation, purification, and pharmacological actions of a novel group of drugs, namely indolealkylamino-withasteroid conjugates, from WS. It is, however, possible that these novel compounds may be obtained from other plants as well.
In view of the above, it would be desirable to provide a potent and therapeutically effective extract of WS in a pharmaceutical or nutraceutical composition having improved properties for the treatment or prevention of ailments, in particular, neurological deficiencies and depression. It would also be desirable to provide an extract of WS for use as a nutritional supplement.
If a way could be found to enhance or enrich the levels of withanolides and/or withasteroids in a WS extract, this would represent a valuable contribution to the art.